The principal aim of this research is to study the mechanism of RNA priming in human mitochondrial (mt) DNA replication. This work will focus on priming at the mt L-strand origin. From previous in vivo studies, we know that the L-strand origin initiates RNA primer formation when during progressive extension of the nascent H-strand, this site becomes single-stranded (SS). To study these events in vitro, I will initially attempt to identify a mt ATPase that selectively recognizes this SS origin sequence. ATPase recognition of the origin sequence may be an obligatory first step in the self-assembly of a functional priming complex. This approach stems from our knowledge of the sequence and potential secondary structure of the L-strand origin and is patterned from studies with E. coli protein n'. Protein n' is a specific ATPase (dATPse) that recognizes the Phi X174 SS, lagging strand origin and guides assembly of an E. coli DNA replicative priming complex, the "primosome". Regardless of whether the search for the mt ATPase succeeds, other mt proteins that can collectively generate a specific RNA primer at the SS L-strand origin will be identified and purified. The capacity of purified DNA polymerase-Gamma to utilize this primer in vitro will be explored. In vitro assays of the ATPase and priming proteins will exploit their dependence on the cloned, L-strand origin templated and inactivity on other SS DNAs. These proteins will each be purified from mt extracts and characterized. Enzyme fractionation will use DNA-cellulose, ion exchange and gel filtration chromatography. Later, monoclonal antibody affinity resins will be generated and used. The RNA primer synthesized in vitro will be sequenced. Also, the cloned origin template will be partially digested, re-cloned, and assayed in vitro to help determine the minimal DNA sequence required for both ATPase activity and priming. In addition, E. coli protein n' will be further characterized and a comparative study with the mt ATPase will be carried out. These studies are relevant not only to biochemistry of DNA replication, but to cell biology and pathology as well. The basic enzymology of DNA initiation and priming must be determined before we can hope to understand the regulation of DNA synthesis and the abberations which characterize malignant cells.